ABSTRACT Anopheles mosquitoes are the primary vectors of malaria, one of the deadliest diseases in human history. New genomic resources and genetic methods provide opportunities to improve our understanding of mosquito biology, which will facilitate the development of novel strategies to control malaria and other mosquito-borne diseases. For example, a better understanding of the molecular mechanism of sex determination and male fertility could improve mosquito control applications that rely on the release of the non-biting males. The Anopheles Y chromosome regulates male determination and mating behavior. In Drosophila, Y chromosome regulates male fertility and global gene expression. Thus, Y chromosome is of critical importance to our understanding of male biology in many taxa including Anopheles. Several Y chromosome genes have been discovered in mosquitoes, including the primary signals or the earliest transcribed Y genes Guy1 and gYG2/YOB in An. stephensi and An. gambiae, respectively. Both Guy1 and gYG2/YOB confers female lethality, consistent with their function as a male-determining factor that also regulates dosage compensation. Although both GUY1 and gYG2/YOB proteins are 56 amino acid long and share predicted secondary structures, there is no significant sequence similarity to support common origin. Y chromosome remains largely unexplored in other Anopheles mosquitoes and only females were sequenced in the 16 Anopheles genome project. There is a tremendous opportunity to improve our understanding of the function and evolution of Y chromosome genes in Anopheles mosquitoes. We will pursue the following Specific Aims: 1) Identify, verify, and characterize Y chromosome genes in eight Anopheles species; 2) Investigate the evolutionary and functional relationship of the primary Y signals in a broad range of Anopheles mosquitoes; and 3) Study the origin and conservation of all other Y chromosome genes in Anopheles mosquitoes. By testing the ability of primary Y chromosome signals from multiple species to confer male bias in transient embryonic assays, this study lays the foundation for future mosquito control applications. By leveraging established methodologies and existing resources from the 16 Anopheles genome project, we will provide a high return on NIH investment.